Vertebrate sensory hair cells are sensitive to damage from a variety of genetic and environmental factors, including exposure to aminoglycoside antibiotics. Exposure to these antibiotics results in death of sensory hair cells. In mammals the loss of hair cells can result in permanent hearing loss and/or balance disturbances. Recent evidence indicates that exposure to aminoglycosides activates an intrinsic cell death pathway in hair cells. This cascade involves activation of a family of cysteine proteases called caspases. Caspase activation is the hallmark of programmed cell death in other systems, and different caspases are activated in response to different cell death-promoting stimuli. The overall goal of the proposed research is to utilize an in vitro model of mammalian sensory hair cell ototoxicity to determine the involvement of specific caspases in aminoglycoside-induced hair cell death. In the first set of experiments, I will determine which caspases are activated in sensory hair cells in response to aminoglycoside exposure and evaluate whether the cells activating caspases are subsequently dying. The second set of experiments will focus on determining which upstream caspases mediate activation of specific downstream caspases in hair cells exposed to aminoglycosides. Such an understanding of the order of caspase activation is an essential step toward understanding the signaling cascade that takes place in a damaged hair cell. The third set of experiments is designed to determine which individual caspases are necessary for aminoglycoside-induced programmed cell death in hair cells. Taken together, the experiments are designed to provide the first examination of the cascade of caspase activation in hair cells beginning with the cell death trigger and proceeding through the actual execution of the cell.